Caffeine fraction obtained from tea leaves and a method for inducing agrobacterium tumefaciens-mediated genetic transformation in plants using said caffeine fraction

ABSTRACT

The present invention relates to a thermolabile caffeine fraction useful for an efficient  Agrobacterium -mediated genetic transformation in plant systems to develop desired traits in plant, and a method of preparing said fraction from tea leaves and also, an efficient and cost-effective method of introducing said  Agrobacterium -mediated genetic transformation into plant systems using said caffeine fraction of tea leaves.

TECHNICAL FIELD

The present invention relates to a thermolabile caffeine fraction usefulfor an efficient Agrobacterium-mediated genetic transformation in plantsystems to develop desired traits in plant, and a method of preparingsaid fraction from tea leaves and also, an efficient and cost-effectivemethod of introducing said Agrobacterium-mediated genetic transformationinto plant systems using said caffeine fraction of tea leaves.

BACKGROUND ART

Agrobacterium is a gram negative soil bacterium that transfers its Tiplasmid or the ‘Tumor inducing plasmid’ into the cells of mostdicotyledonous plants and quite a good number of monocotyledonousplants. The Ti plasmid undergoes cell-cell recognition, signaltransduction, cellular and nuclear import and finally T-DNA integration(Winans S C Two-way chemical signaling in Agrobacterium-plantinteractions Microbiological-Reviews. 1992, 56: 1, 12-31).

The transfer DNA or the T-DNA harbouring the oncogenes bounded by the 25bp imperfect border repeats on either side brings about genetictransformation and hence crown gall disease. The process of signaltransduction is initiated by a set of several ‘virulence genes’, ofwhich 7 genes are the most important.

The first step of signal reception and then transduction is triggered bythe inducers like phenolics and sugars (Ankenbauer R G; Nester E WSugar-mediated induction of Agrobacterium tumefaciens virulence genes:structural specificity and activities of monosaccharides. Journal ofBacteriology. 1990, 172: 11, 6442-6446).

For the past two decades, revolutionary work has been done ondevelopment of genetically transformed plants wherein, the disarmedstrains of Agrobacterium (i. e. the Ti plasmid with the oncogenesreplaced by genes of interest) are employed to produce plants as perones requirements at a much shorter time. The inducers that are used forsuch genetic transformation experiments are generally the Acetosyringone(Morris J W; Morris R O Identification of an Agrobacterium tumefaciensvirulence gene inducer from the Pinaceous gymnosperm Pseudotsugamenziesii. Proceedings of the National Academy of Sciences of the UnitedStates of America 1990, 87: 9, 3614-3618) and the Hydroxy-acetosyringonethat are commercially provided by the Sigma Aldrich Company, USA.

Tea leaves when extracted, are known to have fractions of caffeine,catechins and other flavonols and amino acids. Some chance experimentsindicated that particular fractions of tea containing caffeine arecapable of promoting infection by Agrobacterium, cell-cell recognitionand virulence. This led us to believe that the caffeine fractions can beused as a virulence inducer during genetic transformation experimentsinstead of Acetosyringone or Hydroxy-acetosyringone.

Besides containing high levels of the 6 types of catechins (C) and theirderivatives viz., epicatechin (EC), gallocatechins (GC),epigallocatechins (EGC), epicatechin gallate (ECg), epigallocatechingallate (EGCg), tea leaves also contain caffeine, amino acids,nitrogenous compounds, vitamins, inorganic elements, carbohydrates andlipids (Chu D C and Juneja L. R. General chemical composition of greentea and its infusion. In: Chemistry and Application of Green Tea. 1997.CRC Press, N. York. eds. Yamamoto T., Juneja L. R., Chu D C, Kim M.,pp.).

The report of Sunilkumar et al., 1999. (Sunilkumar G; Vijayachandra K;Veluthambi K 1999, Pre-incubation of cut tobacco leaf explants promotesAgrobacterium-mediated transformation by increasing vir gene induction.Plant Science Limerick. 141: 1, 51-58) indicated that the requirement ofpreincubation for increased transformation efficiency can be obviated bythe addition of 100 μM acetosyringone to the freshly cut leaf ringsduring the co-cultivation with Agrobacterium. The production of vir geneinducers by the leaf rings during the pre-incubation period is animportant factor that contributes to increased transformation efficiencyof Agrobacterium upon pre-incubation but the drawback is that the effectof the inducer ‘acetosyringone’ and pre-incubation is similar and thusinducers do not have much role to play.

Expression of Agrobacterium tumefaciens virulence (vir) genes andtransformation of dicots by this organism are dependent upon host plantphenolic compounds and several alkylsyringamides, syringic acid,synthetic amides like ethylsyringamide, ferulic or sinapic acids arepowerful inducers of vir genes (Vir gene inducing activities ofhydroxycinnamic acid amides in Agrobacterium tumefaciens Berthelot, K;Buret, D; Guerin, B; Delay, D; Negrel, J; Delmotte, F M. Phytochemistry.1998, 49: 6, 1537-1548).

However, none of the inducers tested exhibited higher activity thanacetosyringone, the reference compound for vir gene induction, with theexception to ethylsyringamide at concentrations above 1 mM. When testedon A. tumefaciens strain A348 (pSM243cd), ethylferulamide andethylsinapamide are more efficient than the corresponding phenolic acidsbut only above 100 μM.

The major draw back is that the above mentioned inducers are veryexpensive chemical compounds that are required to be used at highconcentrations above 100 μM. Moreover, the compounds like acetosyringoneare manufactured only by a few select companies like Sigma Aldrich andrequires import from United States of America by research laboratoriessituated in developing countries.

Lee et al., 1995 (Lee, YongWoog; Jin, ShouGuang; Sim, WoongSeop; Nester,E W; Lee, Y W; Jin, S G; Sim, W S Proceedings of the National Academy ofSciences of the United States of America. 1995 Genetic evidence fordirect sensing of phenolic compounds by the VirA protein ofAgrobacterium tumefaciens. 92: 26, 12245-12249.) reported that thevirulence (vir) genes of Agrobacterium tumefaciens are induced bylow-molecular-weight phenolic compounds and monosaccharides through atwo-component regulatory system consisting of the VirA and VirGproteins. The vir-inducing abilities of 15 different phenolic compoundslike acetovanillone was tested using four wild-type strains of A.tumefaciens KU12, C58, A6, and Bo542. By transferring different Tiplasmids into isogenic chromosomal backgrounds, the phenolic-sensingdeterminant was shown to be associated with Ti plasmid. Subcloning of Tiplasmid indicates that the vira locus determines which phenoliccompounds can function as vir gene inducers. These results suggest thatthe VirA protein directly senses the phenolic compounds for vir geneactivation. The drawback of this report is that subcloning of Ti plasmidis required for the identification of the accurate phenolics inducer forvir a locus.

Hess et al., 1991 (Hess, K M; Dudley, M W; Lynn, D G; Joerger, R D;Binns, A N. 1991, Mechanism of phenolic activation of Agrobacteriumvirulence genes: development of a specific inhibitor of bacterialsensor/response systems. Proceedings of the National Academy of Sciencesof the United States of America. 1991, 88: 17, 7854-7858) reported thatthe aglycone of the dihydrodiconiferyl alcohol glycosides were potentinducers of virulence gene expression in A. tumefaciens. Using thismodel, a specific inhibitor of vir induction was developed. The drawbackof this report is that this inhibitor did not affect the induction ofother genes on the Ti plasmid but irreversibly blocks vir expression.

Fortin et al., 1992 (Fortin, C; Nester, E W; Dion, P. 1992, Growthinhibition and loss of virulence in cultures of Agrobacteriumtumefaciens treated with acetosyringone. Journal of Bacteriology. 174:17, 5676-5685) reported that acetosyringone, a phenolic inducer of thevirulence (vir) genes of A. tumefaciens, inhibited the growth of thenopaline type strains T37 and C58 incubated under acidic conditions. Twoother vir inducers, sinapinic acid and syringaldehyde, also inhibitedgrowth and promoted accumulation of avirulent clones in cultures ofstrains C58F and T37. On the other hand, various acetosyringoneanalogues reported not to induce the vir genes did not act as growthinhibitors. Mutants of strain C58F lacked the capacity to induce avirB::lacZ fusion in the presence of acetosyringone. The drawback ofthis report is that while some inducers were promotive, others areinhibitory and are also strain specific.

Delmotte et al., 1991 (Delmotte F M; Delay D; Cizeau J; Guerin B; LepleJ C 1991. Agrobacterium vir-inducing activities of glycosylatedacetosyringone, acetovanillone, syringaldehyde and syringic acidderivatives. Phytochemistry., 30: 11, 3549-3552.) reported that when A.tumefaciens str. A348 (pSM358) harbouring a virE::lacZ fusion plasmidwas used to detect the ability of 13 synthetic acetosyringone,acetovanillone, syringaldehyde and syringic acid beta-glycosides toinduce virulence, the activity of the reporter beta-galactosidase wasdetected by spectrofluorimetry using 4-methylumbelliferylbeta-galactopyranoside as substrate. Acetosyringonylbeta-L-fucopyranoside was the most active monoglycoside tested; even athigh concentrations this compound was devoid of toxic effects. However,monoglycosides were less active vir inducers than free acetosyringone.In contrast, the beta-maltoside of syringaldehyde showed higher activitythan the free phenol at high concentrations. The activity of suchglycosylated inducers may be related to specific sugar receptors on thebacterial cell surface. The drawback of the report is that theacetosyringone is costly compound that need to be imported from SigmaAldrich, USA.

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to develop a thermolabilecaffeine fraction useful for Agrobacterium-mediated genetictransformation in plant.

Another main object of the present invention is to develop athermolabile caffeine fraction from tea leaves.

Yet another object of the present invention is to characterize thethermolabile caffeine fraction obtained from tea leaves.

Still another object of the present invention is to develop a fractionfrom tea leaves with an ability to induce strain non-specificAgrobacterium-mediated genetic transformation in plant

Still another object of the present invention is to develop a naturaland effective inducer for Agrobacterium-mediated genetic transformationin plant.

Still another object of the present invention is to develop aneconomical and cost-effective inducer for Agrobacterium-mediated genetictransformation in plant.

Still another object of the present invention is to determine the idealconcentration range of the chloroform fraction of tea leaf forAgrobacterium-mediated genetic transformation in plant.

Further, another object of the present invention is to develop a methodof preparing a thermolabile caffeine fraction from tea leaves.

Another main object of the present invention is to develop a method tomethod of preparing chloroform fraction from the tea leaf extract havingthe property of inducing Agrobacterium-mediated genetic transformationin plant.

Further, another object of the present invention is to develop anefficient method for introducing said Agrobacterium-mediated genetictransformation into plant.

Another object of the present invention is to develop an efficientmethod for introducing said Agrobacterium-mediated genetictransformation into plant using said caffeine fraction of tea leaves.

Yet another object of the present invention is to develop acost-effective method of introducing said Agrobacterium-mediated genetictransformation into plant.

Still another object of the present invention is to develop a method forintroducing said Agrobacterium-mediated genetic transformation intoplant using naturally occurring source.

Still another object of the present invention is to develop a substitutefor the cost of the transformation inducers like acetosyringone andhydroxy-acetosyringone.

Further, another object of the present invention is to develop a methodfor introducing genes of desired traits in plants byAgrobacterium-mediated genetic transformation into plant using saidcaffeine fraction of tea leaves.

Further, another object of the present invention is to compare thetransformation inducing capability of both autoclaved and filtersterilized caffeine fractions.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to a thermolabile caffeine fraction usefulfor an efficient Agrobacterium-mediated genetic transformation in plantsystems to develop desired traits in plant, and a method of preparingsaid fraction from tea leaves and also, an efficient and cost-effectivemethod of introducing said Agrobacterium-mediated genetic transformationinto plant systems using said caffeine fraction of tea leaves.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Accordingly, the present invention relates to a thermolabile caffeinefraction useful for an efficient Agrobacterium-mediated genetictransformation in plant systems to develop desired traits in plant, anda method of preparing said fraction from tea leaves and also, anefficient and cost-effective method of introducing saidAgrobacterium-mediated genetic transformation into plant systems usingsaid caffeine fraction of tea leaves.

In an embodiment of the present invention, wherein a cost-effective andefficient method of using thermolabile caffeine fraction of tea leavesas a natural inducer for bacteria Agrobacterium tumefaciens mediatedgenetic transformations in plants to produce desired traits in theplants, said method comprising step of:

-   -   inoculating strains of the bacteria into liquid modified Yeast        Mannitol Broth,    -   incubating the inoculum for about 12-16 hrs at about 25-30° C.,        at about 150-200 rpm in dark,    -   harvesting the incubated inoculum at about 0.6-0.8 optical        density at 600 nm for 1×10⁹ cells/ml during log phase of        bacterial growth to obtain pellet,    -   suspending the pellet in fresh Yeast Mannitol Broth without        damaging the bacterial cells to obtain a suspension,    -   immersing explants of different plants in bacterial suspension        for about 5-35 minutes,    -   incubating explants on incubation medium for different periods        of 1-10 days,    -   using caffeine fraction at concentrations of about 0.5-300 μg/ml        in fresh cultures of the bacteria to induce vir genes, thereby        transferring Ti plasmid harbouring the transgene into the plant        for genetically transforming the plants with genes of desired        traits.

In another embodiment of the present invention, wherein pelleting ofliving bacterial cells by centrifugation at 15-30 minutes at 4000-8000rpm and 25-30° C.

In yet another embodiment of the present invention, wherein caffeinefraction is better inducer as compared to commercially availableinducers.

In still another embodiment of the present invention, wherein a methodof preparing thermolabile caffeine fraction from the tea plant, saidmethod comprising steps of:

-   -   extracting dried tea leaves overnight at room temperature with        about 10-40% aqueous acetone,    -   filtrating tea leaves extract with n-hexane to obtain aqueous        and lipid layers,    -   extracting the aqueous layer with petroleum ether and ethyl        acetate to remove catechins from the aqueous layer,    -   extracting aqueous layer of step (c) with chloroform to obtain        chloroform layer,    -   estimating total caffeine fraction concentration in the        chloroform layer,    -   sterilizing caffeine fraction both by autoclaving and filter        sterilization to obtain said fraction.

In still another embodiment of the present invention, wherein athermolabile caffeine fraction.

In another embodiment of the present invention, a thermolabile caffeinefraction of tea leaves-a substitute for acetosyringone for Agrobacteriummediated genetic transformations which comprises step of:

-   (i) inoculation of two strains of Agrobacterium tumefaciens viz.    EHA105 (mild) and GV2260 (virulent) harbouring the antibiotic    resistance genes for kanamycin or hygromycin and the reporter gus    gene from mother cultures of respective strains into 10-30 ml liquid    modified Yeast Mannitol Broth with kanamycin or hygromycin,-   (ii) incubation for 12-16 hrs at 25-30° C. and 150-200 rpm in dark,-   (iii) harvesting at 0.6-0.8 optical density at 600 nm for 1×10⁹    cells/ml during log phase of bacterial growth,-   (iv) pelleting of living bacterial cells by centrifugation at 15-30    minutes at 4000-8000 rpm and 25-30° C.,-   (v) suspension of bacterial pellet in fresh 5-25 ml of Yeast    Mannitol Broth without damaging the bacterial cells,-   (vi) optimization of bacterial cell density at 1×10⁹ cells/ml by    measuring optical density at 600 nm,-   (vii) immersion of various explants of different plants in bacterial    suspension for 5-35 minutes,-   (viii) blotting of explants on filter papers to remove excess    Agrobacterium tumefaciens,-   (ix) incubation of explants on incubation medium for different    periods of 1-10 days,-   (x) 100-500 g fresh tea leaves of Kangra jat dried in an oven at    60° C. to a constant weight,-   (xi) dried leaves extracted overnight at room temperature with    0.4-1.2 litres of 10-40% aqueous acetone and filtered,-   (xii) the filterate extracted with 100-500 ml of n-hexane to obtain    two layers for removal of lipids,-   (xiii) the aqueous layer taken and extracted with petroleum ether    (100-300 ml) and ethyl acetate (100-400 ml) to remove catechins,-   (xiv) aqueous layer taken and extracted with chloroform (100-400 ml)    and ammonia solution (3-10%),-   (xv) chloroform layer concentrated to 10-50 ml and total caffeine    estimated,-   (xvi) concentrated chloroform layer serves as a caffeine fraction,-   (xvii) sterilization of caffeine fraction both by autoclaving and    filter sterilization-   (xviii) use of caffeine fraction at concentrations of 0.5-300 μg/ml    in fresh cultures of Agrobacterium tumefaciens instead of    acetosyringone.-   (xix) transfer of various explants of different plants to    regeneration medium containing different concentrations of 0.5-300    μg/ml caffeine fractions for inducing the vir genes and increasing    the transformation efficiency.-   (xx) transfer of Agrobacterium tumefaciens free explants to    regeneration medium containing selection antibiotics for further    regeneration and transgenic plant development, and-   (xxi) comparative analysis of the caffeine fraction (both autoclave    and filter sterilized-please refer FIGS. 1 and 2) and catechin    fraction and the crude tea extract though HPLC using acetonitrile    and phosphoric acid in gradient mode (0.05 to 0.2%) for detection of    virulence inducing compound using diode array detector at 250 to 280    nm wavelength as shown in Table 1 and 2 here below.

TABLE 1 filter sterilized caffeine fraction Conc (μg/ml) 12 hrs 14 hrs18 hrs 20 hrs C0 0.377 0.377 0.377 0.393 C50 0.36 0.396 0.416 0.409 C1000.352 0.39 0.423 0.415

TABLE 2 autoclaved caffeine fraction Conc (μg/ml) 0 hr 6 hrs 12 hrs 18hrs 24 hrs C0 0.1 0.37 0.42 0.443 0.445 C50 0.104 0.367 0.429 0.4230.402 C100 0.109 0.371 0.44 0.428 0.432

In still another embodiment of the present invention, genetictransformation in plants via Agrobacterium is brought about by inductionof virulence or vir genes and transfer of the Ti plasmid harbouring thetransgene into the plant tissue. Transgenes that code for desirabletraits like disease and stress resistance, crop quality, flavors,colors, better shelf life when introduced into the plant tissues canbring about crop improvement in a shorter time than conventionalbreeding. Generally phenolic inducers are responsible for the inductionof the vir genes and the popular commercially available inducers are theacetosyringone or the hydroxy-acetosyringone. These commerciallyavailable inducers are not only expensive but are also confined to fewselect companies like Sigma Aldrich, USA.

In still another embodiment of the present invention, caffeine fractionof tea leaves has the potential for substituting these commerciallyavailable inducers as they can induce the vir genes and bring aboutgenetic transformation. This natural fraction that is easily availableis not only cost effective but can also improve the transformationefficiency.

In another embodiment of the present invention, the virulence inducercaffeine fraction from tea leaves can be used for transgenic productionusing different explants of different plant species.

In still another embodiment of the present invention, differentconcentrations of filter sterilized caffeine fractions of tea leaveswere added as above.

In still another embodiment of the present invention, differentconcentrations of autoclaved caffeine fractions of tea leaves were addedas above.

In still another embodiment of the present invention, Clostridiumperfringens, a foodborne pathogen, exerts its toxic effect on humanswhen producing a toxin during sporulation in the intestine.Investigations were undertaken to determine the effect of caffeine whichis consumed in large amounts worldwide in the diet on sporulation andsubsequent toxin formation. In the presence of 100 μg caffeine/ml or 200μg theobromine/ml, sporulation of C. perfringens NCTC 8679 rose from <1to 80 or 85%. Enterotoxin concentration increased from undetectablelevels to 450 μg/mg of cell extract protein. Heat-resistant spore levelsincreased from <1000 to between 1×107 and 2×107/ml. Caffeine caused a 3-to 4-fold increase in the percentages of cells possessing retractilespores and a similar increase in enterotoxin concentration.

In still another embodiment of the present invention, the study onmechanism by which caffeine stimulates sporulation of C. perfringensindicated that cultures exposed to caffeine had significantly elevatedlevels of intracellular adenosine and guanosine triphosphates (Nolan LL; Labbe R G; Craker L E (ed.); Nolan L (ed.); Shetty K Effect of plantalkaloids on the sporulation of a food-borne pathogen. Internationalsymposium on medicinal and aromatic plants, Amherst, Mass., USA, 27-30Aug. 1995. Acta-Horticulturae. 1996, No. 426, 287-295).This property ledus to employ the caffeine fraction from tea leaves for inducing ofvirulence genes in Agrobacterium mediated genetic transformations andalso a substitute of an expensive chemical acetosyringone. Moreover,since the the virulence inducer caffeine fraction from tea leaves arealso obtained from the maintenance foliage leaves that are generallydiscarded and burnt, this method appears to be all the more economicaland cost effective. Use of the virulence inducer caffeine fraction fromtea leaves is specially important in increasing the transformationefficiency.

In still another embodiment of the present invention, a thermolabilecaffeine fraction of tea leaves—a substitute of acetosyringone forAgrobacterium mediated genetic transformations. (see FIG. 4)

In still another embodiment of the present invention, a thermolabilecaffeine fraction of tea leaves—a substitute for acetosyringone forAgrobacterium mediated genetic transformations obviates the drawbacks asdetailed above.

In still another embodiment of the present invention, the novelty ofthis method is that a cost effective natural extract was identified thatcould substitute for the cost of the inducers like acetosyringone andhydroxy-acetosyringone.

In still another embodiment of the present invention, the caffeinefraction of tea leaves can be used for Agrobacterium tumefaciensmediated genetic transformation of different systems or plants orexplants.

In still another embodiment of the present invention, an economicalsystem for obtaining an inducer without involving any costly extractionmethods wherein the crude leaf extract with bactericidal activity isalso extracted without requiring expensive instruments.

In still another embodiment of the present invention, the caffeinefraction of tea leaf with virulence inducer activity can be obtained inabundance from naturally growing tea bushes.

In still another embodiment of the present invention, the virulenceinducer caffeine fraction from tea leaves is of natural origin and isnot of synthetic or semi-synthetic nature.

In still another embodiment of the present invention, the virulenceinducer caffeine fraction from tea leaves can be obtained in abundancefrom actively growing apical shoots i. e. two and a bud making iteconomical.

In still another embodiment of the present invention, the virulenceinducer caffeine fraction from tea leaves can be obtained in abundanceeven from the lower maintenance foliage thereby making it moreeconomical as generally such leaves are discarded or burnt.

In still another embodiment of the present invention, the crude extractcan also be used during ‘hairy root production’ using Agrobacteriumrhizogenesis.

In still another embodiment of the present invention, the transformationefficiency by Agrobacterium can be increased considerably.

In still another embodiment of the present invention, use of tea leafextract for Agrobacterium tumefaciens genetic transformation indifferent systems or plants or explants wherein the generally usedsynthetic inducers are ineffective.

In still another embodiment of the present invention, deriving a costeffective system for Agrobacterium tumefaciens genetic transformationwherein no commercial and yet costly inducers are used.

The crude tea leaf extract in patent no NF 14/02 was obtained by usingacetone and n-hexane where as the caffeine fraction in patent no NF221/02 was obtained by using solvent extraction using ethyl acetate andpetroleum benzene for removal of catechins. The crude tea leaf extractcontained generally catechin and caffeine whereas the caffeine fractionhad only caffeine, therefore they should not be treated as one. Thesupporting data in FIG. 3 represents: Effect of different fractions oftea extract on the growth of Agrobacterium tumefaciens culture. Thisclearly substantiates the difference between both the fractions.

Use of caffeine fraction at concentrations of 0.5-300 μg/ml in themedium of fresh cultures of Agrobacterium tumefaciens instead ofacetosyringone. This refers to the concentration of caffeine in theculture medium and actually refers to concentrations ranging from0.5-300 μg of caffeine in the medium. The concentration of the induceris indirectly estimated as the concentration of caffeine in thefraction.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

FIG. 1 shows effect of filter sterilized and autoclaved caffeine teafraction on the growth of Agrobacterium tumefaciens culture.

FIG. 2 shows comparison of filter sterilized and autoclaved caffeinefractions.

FIG. 3 shows effect of different fractions of tea extract on the growthof Agrobacterium tumefaciens culture.

FIG. 4 shows comparison of the effect of acetosyringone and the caffeinefraction on the growth of Agrobacterium tumefaciens.

The following examples are given by way of illustration and thereforeshould not be construed to limit the scope of the present invention.Example 1

Two strains of Agrobacterium tumefaciens viz. EHA105 (mild) and GV2260(virulent) harbouring the antibiotic resistance genes for kanamycin orhygromycin and the reporter gus gene from mother cultures of respectivestrains were inoculated into 10-30 ml liquid modified Yeast MannitolBroth with kanamycin or hygromycin and incubated for 12-16 hrs at 25-30°C. and 150-200 rpm in dark. Cells were harvested at 0.6-0.8 opticaldensity at 600 nm for 1×10⁹ cells/ml during log phase of bacterialgrowth. Living bacterial cells were pelleted of by centrifugation at15-30 minutes at 4000-8000 rpm and 25-30° C. Bacterial pellet wassuspended in fresh 5-25 ml of Yeast Mannitol Broth without damaging thebacterial cells and cell density was optimized at 1×10⁹ cells/ml bymeasuring optical density at 600 nm. Various explants of differentplants were immersed in bacterial suspension for 5-35 minutes. Explantswere blotted on filter papers to remove excess Agrobacteriumtumefaciensis. Explants incubated on incubation medium for differentperiods of 1-10 days. Fresh tea leaves (100-500 g) of Kangra jat driedin an oven at 60° C. to a constant weight and extracted overnight atroom temperature with 0.4-1.2 litres of 10-40% aqueous acetone andfiltered. The filtrate extracted with 100-500 ml of n-hexane to obtaintwo layers for removing lipids. The aqueous layer was extracted withpetroleum ether (100-300 ml) and ethyl acetate (100-400 ml) to removecatechins. The aqueous layer was taken and extracted with chloroform(100-400 ml) and ammonia solution (3-10%). The chloroform layer wasconcentrated to 10-50 ml and total caffeine was estimated. Theconcentrated chloroform layer served as a caffeine fraction and wassterilized both by autoclaving and filter sterilization method. Theconcentrated chloroform layer served as a caffeine fraction which wasused at concentrations of 0.5-300 μg/ml in fresh cultures ofAgrobacterium tumefaciens. Various explants of different plants weretransferred to regeneration medium containing different concentrationsof 0.5-300 μg/ml caffeine fractions for inducing the vir genes andincreasing the transformation efficiency.

Agrobacterium tumefaciens free explants were transferred to regenerationmedium containing selection antibiotics for further regeneration andtransgenic plant development. Comparative analysis of the threefractions viz. the caffeine fraction (both autoclave and filtersterilized), the catechin fraction and the crude tea extract thoughHPLC. Acetonitrile and phosphoric acid (0.05 to 0.2%) were used ingradient mode for detection of ‘virulence inducing compound’ with thehelp of diode array detector at 250 to 280 nm wavelength.

Example 2

Caffeine fractions were filter sterilized and used as an inducing agentinstead of acetosyringone in different explants for Agrobacteriumtumefaciens mediated transformations as described above in Example-1.

Example 3

Caffeine fractions were autoclaved used as an inducing agent instead ofacetosyringone in different explants for Agrobacterium tumefaciensmediated transformations as described above in Example-1 and 2.

The Main Advantages of the Present Invention are:

-   (1) Caffeine fraction of tea leaf can be used as a potent virulence    inducing agent for Agrobacterium tumefaciens mediated genetic    transformations instead of acetosyringone.-   (2) Caffeine fraction of tea leaf can be used for Agrobacterium    tumefaciens mediated genetic transformation in different systems or    plants or explants wherein the commercially used virulence inducing    agent acetosyringone is ineffective.-   (3) Since no commercial virulence inducer need to be used, caffeine    fraction of tea leaf prove to be a cost effective system for    Agrobacterium tumefaciens mediated genetic transformation.-   (4) Since the caffeine fraction of tea leaf with virulence activity    is obtained without involving any expensive instruments or costly    extraction methods, its use proves to be an economical method.-   (5) The caffeine fraction of tea leaf with virulence inducing    ability can be easily obtained in abundance from naturally growing    tea bushes round the year.-   (6) The caffeine fraction of tea leaf with virulence inducing    ability is of natural origin and is not of synthetic or    semi-synthetic nature.-   (7) The caffeine fraction of tea leaf with virulence inducing    ability can be obtained in abundance from even the lower maintenance    foliage thereby, making it more economical as generally such leaves    are discarded or burnt.-   (8) The caffeine fraction of tea leaf can also be used as an agent    with virulence inducing ability in different in vitro systems    involving different plants or explants.-   (9) The caffeine fraction of tea leaf with virulence inducing    ability can also be used during ‘hairy root production’ using    Agrobacterium rhizogenesis.-   (10) The caffeine fraction of tea leaf with virulence inducing    ability can be used to increase the efficiency of Agrobacterium    mediated transformation.

1-3. (canceled)
 4. A method of preparing thermolabile caffeine fractionfrom the tea plant, said method comprising steps of: a. extracting driedtea leaves overnight at room temperature with about 10-40% aqueousacetone, b. filtrating tea leaves extract with n-hexane to obtainaqueous and lipid layers, c. extracting the aqueous layer with petroleumether and ethyl acetate to remove catechins from the aqueous layer, d.extracting aqueous layer of step (c) with chloroform to obtainchloroform layer, e. estimating total caffeine fraction concentration inthe chloroform layer, f. sterilizing caffeine fraction both byautoclaving and filter sterilization to obtain said fraction. 5.(canceled)